NIH-3T3 Fibroblasts (National Collection of Authenticated Cell Cultures, China) were cultured in DMEM medium (high glucose, BBI) with 10% fetal bovine serum (FBS, Biological Industries) and 1% antibiotics/antimycotics (BBI) at 37 ​°C under 5% CO2 concentration. Cells were passaged at 70–90% confluency and plated at a density of 20%. After culturing the cells on hydrogels for 12–36 ​h, SPY555-Actin (1 ​μM) were added for actin staining and the cells were further incubated for 12 ​h in the above imaging medium [48]. The cells were then directly imaged using Nikon microscope. The pEGFP vinculin was a gift from Kenneth Yamada (RRID: Addgene_50,513). The jetOPTIMUS® transfection reagent kit (Polyplus-transfection SA, Illkirch, France) was used to transfer plasmids into cells as described by the manufacturer's protocol. All procedures are based on a standard protocol provided by Polyplus-transfection SA and incubated for 24–48 h before imaging.

The active forces exerted from the extracellular matrix (ECM) to mechanoreceptors have crucial impact on many cell functions and disease development. However, our understanding of the underlying mechanisms is held back due to the lack of ECM mimicking platform able to apply molecularly resolved forces to cells. Herein, we present novel hydrogel platform capable of generate pN range forces to specific cellular receptors, at molecular scale. This capability was achieved through near-infrared (NIR) light regulated macromolecular actuators functionalized within the platform. This platform enables us to reveal cell responses to molecularly resolved forces under controlled magnitude (150-400 ​pN) and frequency (up to 100 ​Hz) on matrix with varied stiffness. We find the stiffness of the matrix has a large influence on the applied force transduction to cells. This versatile platform holds the potential for establishing correlation between receptor signaling pathways and cellular responses closer to physiological conditions.